Overview

  • Product name
  • Description
    Rabbit polyclonal to Citrulline
  • Specificity
    Using conjugated citrulline glutaraldehyde Protein, antibody specificity was performed with an ELISA test by competition experiments with the following compounds:
    CompoundCross-reactivity ratio
    Citrulline-G-BSA1
    Arginine-G-BSA, Glutamine-G-BSA, Ornithine-G-BSA1 > 100,000
    Conjuaged Citrulline and conjugated Homo-citrulline1:1000
    G = Glutaraldehyde, BSA = Bovine Serum Albumin
  • Tested applications
    Suitable for: ICC/IF, IHC-P, ELISA, IHC-Frmore details
  • Immunogen

    Chemical/ Small Molecule by a Glutaraldehyde linker.

  • General notes
    This polyclonal antibody recognizes only citrulline residues on proteins not free citrulline.

Properties

Applications

Our Abpromise guarantee covers the use of ab6464 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
ELISA 1/5000 - 1/10000.
IHC-Fr 1/1000 - 1/5000.

Target

  • Relevance
    The amino acid Citrulline is required to detoxify the liver from ammonia, which is a waste product of the body from oxidation. Citrulline promotes energy and assists with the immune system. This unusual amino acid is formed in the urea cycle by the addition of carbon dioxide and ammonia to ornithine. It is then combined with aspartic acid to form arginosuccinic acid, which later is metabolized into the amino acid arginine.

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of breast DCIS tumor with apoptotic center labeling Citrulline  with ab6464 at 1/100 dilution. Tissue was fixed in 10% buffered formalin for 24 hrs then transferred to 70% ethanol until processed and embedded and permeabilized with Tris Buffered Saline with 0.025% Triton X-100. Heat mediated antigen retrieval was performed using 10mM Sodium Citrate Buffer with 0.05% Tween 20 pH 6.0. Tissue was blocked with TBS with 10% normal (secondary species) serum and 1% BSA for 1 hour at 37°C, followed by incubation with Anti-Citrulline antibody (ab6464) in TBS with 1% BSA for 18 hours at 4°C at 1/100 dilution. A polyclonal goat anti rabbit IgG biotin conjugate secondary antibody was used at 1/200 dilution. 

     

    See Abreview

  • Immunofluorescence analysis of THP-1 cells, staining Citrulline with ab6464.

    Cells were fixed with paraformaldehyde and blocked with 0.05% BSA for 1 hour at 37°C. Samples were incubated with primary antibody (1/500 in diluent) for 2 hours at 37°C. ab6717 Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (FITC) was used to detect staining.

    See Abreview

  • Competitive curve of the reactivity of ab6464 with conjuaged Citrulline (blue) and conjugated Homo-citrulline (red). The cross-reactivity ratio is around 1:1000.

References

This product has been referenced in:
  • Horibata S  et al. Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration. BMC Cancer 17:378 (2017). Read more (PubMed: 28549415) »
  • Li G  et al. Peptidylarginine Deiminase 3 (PAD3) Is Upregulated by Prolactin Stimulation of CID-9 Cells and Expressed in the Lactating Mouse Mammary Gland. PLoS One 11:e0147503 (2016). Read more (PubMed: 26799659) »

See all 8 Publications for this product

Customer reviews and Q&As

Application
Western blot
Sample
Human Recombinant protein (Histone H3)
Gel Running Conditions
Non-reduced Denaturing
Loading amount
1 µg
Treatment
1µg PAD4 for 24 hrs
Specification
Histone H3
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Mona Biermann

Verified customer

Submitted Sep 26 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 37°C
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10mM Sodium Citrate Buffer with 0.05% Tween 20 pH 6.0
Sample
Human Tissue sections (Breast, DCIS tumor with apoptotic center)
Specification
Breast, DCIS tumor with apoptotic center
Permeabilization
Yes - Tris Buffered Saline with 0.025% Triton X-100
Fixative
Formaldehyde
Username

Ms. Lynne Anguish

Verified customer

Submitted Jun 09 2014

Thank you for contacting us. We currently have two antibodies that will detect the citrulline modification on any proteins: ab6464 and ab100932. While ab6464 has been validated in IHC on frozen tissue sections, we have not tested this antibody for use ...

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Thank you for your reply and this complement of information.

ab6464 has not been tested in Western Blot and may not be suitable for this application. This would explain why you don't see any staining with this antibody.
The information on ...

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Thank you for contacting us.

The source of the antibody informed us that they have never tested the antibody by coating plates with fibrinogen.
Their testing are made using BSA and citrunilated BSA. However, the testing conditions are very...

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Thank you for your email.

I am very happy to hear that the protocol optimizations improved the results and the antibody is now working fine.

I wish all the best for future experiments and research.

Thank you for your inquiry! We are well, thanks- Ihope you are well too!

The two products are from two different laboratories and therefore productions. They are both against bound citrulline and guaranteed by us in ICC/IF, ELISA, IHC-Fr for ...

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Thank you very much for filling in the questionnaire.

The lab is using the belowprotocol to test the antibody. I noticed there are differences, particular concerning the buffers. The positive control is Citrulline-BSA.

Maybe this pr...

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Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Skin)
Specification
Skin
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: EDTA pH 9.0
Permeabilization
No
Blocking step
Casein as blocking agent for 15 minute(s) · Concentration: 0.25% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Jun 15 2012

Thank you for contacting us.

The antibody detects citrullinated proteins. And a chemical modification of the citrulline is not necessary or required. An example of a protein modified with citrulline that comes to mind, is histone H3.
<...

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